Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory–secretory proteins and their biochemical properties

• Published in: Parasitology research (1987) 2014-08, Vol.113 (8), p.2765-2776
• Main Authors: Lee, Jinyoung, Kim, Jong-Hyun, Sohn, Hae-Jin, Yang, Hee-Jong, Na, Byoung-Kuk, Chwae, Yong-Joon, Park, Sun, Kim, Kyongmin, Shin, Ho-Joon
• Format: Article
• Language: English
• Subjects: albumins; Amino Acid Sequence; amino acids; Amoeba; antibodies; Base Sequence; Biomedical and Life Sciences; Biomedicine; cathepsin B; Cathepsin B - genetics; Cathepsin B - metabolism; Cathepsins; cDNA libraries; Cloning, Molecular; collagen; complementary DNA; Cysteine Proteases - genetics; Cysteine Proteases - metabolism; death; encystment; Enzyme Stability; Escherichia coli; excystation; fibronectins; Gene Library; genes; Health aspects; hemoglobin; humans; immune evasion; immunoglobulin A; immunoglobulin G; immunoglobulin M; Immunology; laboratory animals; Medical Microbiology; meningoencephalitis; Microbiology; Molecular Sequence Data; Naegleria fowleri; Naegleria fowleri - enzymology; Naegleria fowleri - genetics; nutrient uptake; Original Paper; parasites; pathogens; polyacrylamide gel electrophoresis; Proteases; protein synthesis; Proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Protozoan - genetics; sodium dodecyl sulfate; Substrate Specificity; Western blotting
• ISSN: 0932-0113; 1432-1955
• DOI: 10.1007/s00436-014-3936-3

Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7–14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.